Project Summary Drug abuse directly contributes to one-third of all HIV infections in the United States. Epidemiological data have demonstrated that opioid abuse is a significant risk factor for HIV infection and progression to AIDS while accumulating evidence reveals possible synergistic interactions between the mu opioid (MOR) and CCR5 chemokine receptors in this pathologic process. Therefore, a thorough understanding of the neural pathways likely involved in opioid enhancement of HIV infection is essential. The putative dimerization of the mu opioid and CCR5 receptors uniquely affects immune cell function and their molecular interactions may underlie their apparently synergistic effects in the CNS. Bivalent ligands have been shown to be powerful molecular tools for characterization of G-protein coupled receptor (GPCR) protein-protein interactions, to interfere with normal function related to these interactions, or even to treat diseases by targeting such interactions. As a proof-of- concept, our recently developed bivalent ligand carrying MOR-CCR5 dual antagonist pharmacophores has shown significantly higher inhibitory effect on HIV-1 invasion in human macrophages and astrocytes compared to a simple mixture of the two antagonists. Our hypothesis is that bivalent ligands containing both an MOR antagonist and a CCR5 antagonist may serve as chemical probes to study the interaction of these receptors with respect to HIV infection enhanced by opioid abuse. We believe a ligand of this kind may serve as a pharmacological probe to help clarify the molecular mechanism of opioid abuse enhanced HIV infection, and help establish this evolving protein-protein interaction model as a potential target for therapeutic intervention in opioid abuse enhanced neuroAIDS. The specific aims of this proposal are to: 1) design and synthesize novel bivalent ligands containing both an MOR antagonist and a CCR5 antagonist as dual pharmacophores by applying crystal structures of ligand bound MOR and CCR5 proteins, molecular modeling, and chemical synthesis; 2) characterize these bivalent ligands with receptor binding and functional assays; and 3) examine the relative efficacy of bivalent ligands in blocking HIV entry and infectivity via CCR5, and CCR5-MOR interactions. We believe such an effort will build the foundation to understand such a complicated biological process, define a novel therapeutic target to treat neuroAIDS, and facilitate future treatment development for the disease.